Nanofibers comprising fibroin as well as system comprising hydrogel and said nanofibers

Abstract

Described herein are nanofibers including an outer membrane and a core, wherein the outer membrane is made of fibroin and the core is a biocompatible and biodegradable polymer. Also described herein are a method for obtaining the nanofibers and the use thereof to convey bioactive molecules and/or particles and/or cells and/or in the treatment of diseases. Also described herein are powdered nanofibers, optionally suspended in an aqueous solution, a hydrogel system including the powdered nanofibers, and the use of the powdered nanofibers and of the hydrogel system to convey bioactive molecules and/or particles and/or cells and/or in the treatment of diseases.

What is claimed is: 
     
         1 . Nanofibers comprising an outer membrane and a core, wherein said outer membrane is made of fibroin and said core is a biocompatible and biodegradable polymer. 
     
     
         2 . The nanofibers according to  claim 1 , wherein said fibroin is the natural or recombinant protein of silk. 
     
     
         3 . The nanofibers according to  claim 1 , wherein said biocompatible and biodegradable polymer is a natural or synthetic polymer, used alone or in combination. 
     
     
         4 . The nanofibers according to  claim 1 , wherein said biocompatible and biodegradable polymer is a water-soluble polymer. 
     
     
         5 . The nanofibers according to  claim 1 , wherein said biocompatible and biodegradable polymer is one or more polymers selected from the group comprising polyethylene oxide (PEO), polycaprolactone (PCL), hyaluronic acid, gelatin, collagen, chitosan, alginate, albumin. 
     
     
         6 . The nanofibers according to  claim 1 , wherein said biocompatible and biodegradable polymer is PEO. 
     
     
         7 . The nanofibers according to  claim 1 , wherein said nanofibers have a diameter from 50 to 2000 nm. 
     
     
         8 . The nanofibers according to  claim 7 , wherein said nanofibers have a diameter from 200 to 500 nm. 
     
     
         9 . The nanofibers according to  claim 1 , wherein a thickness of said outer membrane is from 10 to 750 nm, or from 20 to 250 nm. 
     
     
         10 . The nanofibers according to  claim 1 , wherein said outer membrane has a thickness of 200 nm and said nanofiber has a diameter of 700 nm, or an outer membrane ( 2 ) with a thickness of 100 nm and a diameter of 300 nm, an outer membrane ( 2 ) with a thickness of 100 nm and a diameter of 400 nm, or an outer membrane ( 2 ) with a thickness of 60 nm and a diameter of 250 nm. 
     
     
         11 . The nanofibers according to  claim 1 , wherein said core extends longitudinally. 
     
     
         12 . The nanofibers according to  claim 1 , wherein said core comprises one or more bioactive molecules and/or particles and/or cells. 
     
     
         13 . The nanofibers according to  claim 12 , wherein said bioactive molecules are selected from the group comprising anti-tumor, anti-coagulant, anti-thrombotic compounds, antibodies, vaccines, antibiotics, antivirals, anti-inflammatories, amino acids, peptides, proteins, enzymes, growth factors, angiogenic factors, nucleic acids, salts, fibronectin, glycosaminoglycans, polysaccharides, vitamins, anti-oxidants, anti-microbials. 
     
     
         14 . The nanofibers according to  claim 12 , wherein said bioactive molecules are selected from the group comprising anti-tumor compounds including alkylating agents comprising cyclophosphamide, ifosfamide, chlorambucil, melphalan, estramustine, lomustine, carmustine, carboplatin, cisplatin, oxaliplatin, busulfan, treosulfan, tiotepa, dacarbazine, procarbazine, temozolomide; antimetabolites comprising methotrexate, raltitrexed, pemetrexed, fluorouracil, capecitabine, cytarabine, gemcitabine, tegafur, fludarabine, cladribine, mercaptopurine, thioguanine, pentostatin, clofarabine, nelarabine; cytotoxic antibiotics comprising daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, amsacrine, bleomycin, dactinomycin, mitomycin; derivatives of natural origin comprising paclitaxel, docetaxel, vinblastine, vincristine, vindesine, vinorelbine, irinotecan, topotecan, trabectedin, etoposide, trabectedin; hormones and antagonists comprising diethylstilbestrol, ethinylestradiol, medroxyprogesterone, megestrol, norethisterone, gosereline, leuprorelin, triptorelin, lanreotide, octreotide, tamoxifen, toremifene, fulvestrant, cyproterone, flutamide, bicalutamide, anastrozole, letrozole, exemestane; protein kinase inhibitors comprising dasatinib, erlotinib, imatinib, nilotinib, sunitinib, sorafenib; and monoclonal antibodies comprising panitumumab, trastuzumab, rituximab, alemtuzumab, bevacizumab. 
     
     
         15 . The nanofibers according to  claim 12 , wherein said bioactive molecules are selected from the group comprising small hydrophilic molecules. 
     
     
         16 . The nanofibers according to  claim 15 , wherein said small molecules belong to the group of antibiotics with cytotoxic action including Mitomycin C, Doxorubicin, Epirubicin, and Gemcitabine. 
     
     
         17 . The nanofibers according to  claim 12 , wherein said particles are of inorganic type, organic type, and organic-inorganic hybrids. 
     
     
         18 . The nanofibers according to  claim 12 , wherein said cells are stem cells, primary or line cells, in the native or engineered state. 
     
     
         19 . A method for obtaining nanofibers according to  claim 1 , wherein said method comprises the following steps, not necessarily in the indicated sequence:
 a) providing a fibroin solution at a concentration from 2 to 14% w/w in formic acid;   b) providing a water-soluble polymer solution in an aqueous solution;   c) optionally, adding said bioactive molecules and/or particles and/or cells in said aqueous solution;   d) filling at least two syringes, at least a first syringe with said fibroin solution and at least a second syringe with said water-soluble polymer solution;   e) connecting the syringes to a pumping system of a coaxial electrospinning system; and   f) starting an electrospinning process.   
     
     
         20 . The method according to  claim 19 , wherein said fibroin solution is at a concentration from 2 to 14%, or from 5 to 12%, or from 6 to 12%, or from 5 to 10%, or from 7% to 10%, or from 7 to 9% w/w in formic acid. 
     
     
         21 . The method according to  claim 19 , wherein said aqueous solution is a saline or a culture medium, an enriched aqueous solution, or water. 
     
     
         22 . The method according to  claim 19 , wherein said electrospinning process is performed by setting parameters in the following ranges:
 Flow rate: from 0.1 to 10 ml/h;   Working distance between spinneret (cathode) and metal collector (anode): from 5 to 100 cm;   Potential difference between said cathode and said anode: from 5 to 100 kV.   
     
     
         23 . The method according to  claim 22 , wherein said cathode has a voltage of −10 kV and said anode of +10 kV, or said cathode −5 kV and said anode +15 kV, or said cathode −1 kV and said anode +19 kV, or said cathode 0 kV and said anode +24 kV. 
     
     
         24 . The method according to  claim 19 , wherein said electrospinning process is carried out by working under a hood with activated suction, with a metal collector coated with a removable layer on which the electrospun fibers do not adhere, including an aluminum sheet, a polymeric membrane, or a fabric. 
     
     
         25 . The method according to  claim 19  comprising:
 providing said fibroin solution at a concentration from 6 to 12% w/w in pure formic acid; 
 providing said water-soluble polymer solution as a PEO solution at a concentration from 2 to 6% w/w in water; 
 optionally, dissolving one or more small hydrophilic molecules in said water; 
 filling two syringes with said solutions and connecting them to the pumping system of the coaxial electrospinning system; 
 starting the electrospinning process by setting the parameters in the following ranges: Flow rate: from 1 to 0.5 ml/h, Working distance between spinneret (cathode) and metal collector (anode): from 17 to 19 cm, Applied voltage: keeping the anode at 0 kV, the cathode is brought to 22-26 kV. 
 
     
     
         26 . The method according to  claim 19 , further comprising a product stabilization step. 
     
     
         27 . The method according to  claim 26 , wherein said product stabilization step is a chemical treatment comprising soaking the product obtained by electrospinning in an alcohol and/or water bath for at least 10 minutes. 
     
     
         28 . The method according to  claim 27 , wherein in said product stabilization step, said alcohol is selected from methanol and ethanol, wherein in said bath, said ethanol is from 50 to 100% v/v. 
     
     
         29 . The method according to  claim 27 , wherein in said product stabilization step, said soaking is prolonged for a time of 20-90 minutes. 
     
     
         30 . The method according to  claim 27 , wherein said product is soaked in methanol for about 30 minutes. 
     
     
         31 . Nanofibers according to  claim 1 , which are powdered and have a particle size from 1 to 5000 microns. 
     
     
         32 . An aqueous solution comprising powdered nanofibers according to  claim 31 . 
     
     
         33 . A system that is a hydrogel comprising powdered nanofibers according to  claim 31 . 
     
     
         34 . The system according to  claim 33 , wherein said powdered nanofibers are in a concentration from 0.01% to 5% w/v, or from 0.01% to 1% w/v, or from 0.1 to 0.8% w/v, or from 0.2 to 0.5% w/v. 
     
     
         35 . The system according to  claim 33 , wherein said hydrogel comprises hyaluronic acid+chondroitin sulfate. 
     
     
         36 . The system according to  claim 33 , wherein said hydrogel comprises one or more bioactive molecules and/or particles and/or cells. 
     
     
         37 . The system according to  claim 36 , wherein said one or more bioactive molecules are selected from the group comprising anti-tumor, anti-coagulant, anti-thrombotic compounds, antibodies, vaccines, antibiotics, antivirals, anti-inflammatories, amino acids, peptides, proteins, enzymes, growth factors, angiogenic factors, nucleic acids, salts, fibronectin, glycosaminoglycans, polysaccharides, vitamins, anti-oxidants, and anti-microbials. 
     
     
         38 . The system according to  claim 36 , wherein said nanofibers and/or said hydrogel comprise an active ingredient selected from the group comprising: Mitomycin C, Doxorubicin, Epirubicin, and Gemcitabine. 
     
     
         39 . A method for obtaining a system according to  claim 33 , said method comprising:
 providing nanofibers comprising an outer membrane and a core wherein said outer membrane is made of fibroin and said core is a biocompatible and biodegradable polymer;   grinding said nanofibers to obtain a powder having a particle size from 1 to 5000 microns;   optionally, re-crystallizing; and   dispersing in the hydrogel, optionally added with one or more bioactive molecules and/or particles and/or cells.   
     
     
         40 . A method of regenerating tissues in humans or animals, said method comprising administering the nanofibers according to  claim 1  or a hydrogel containing said nanofibers. 
     
     
         41 . The method according to  claim 40 , wherein said nanofibers and/or said hydrogel are carriers for bioactive molecules and/or particles and/or cells. 
     
     
         42 . (canceled) 
     
     
         43 . A method of treating humans or animals, said method comprising administering the nanofibers according to  claim 1  or a hydrogel containing said nanofibers for the containment of hernias and prolapses, reconstructive prostheses; or as a scaffold for the regeneration of tissues and organs of the peripheral nervous system, the vascular system, including veins, arteries, arterio-venous fistulas for vascular accesses, the lymphatic system, including lymphatic circulatory system, lymph nodes, the cardiovascular system, including coronary arteries and cardiac muscle, the central nervous system, including spinal cord, the skin and layers thereof, the tissues for containing and protecting internal organs, including dura mater, pericardium, pleura, peritoneum, and the tissues of the muscular-skeletal system, including tendons, ligaments, muscles, bones, cartilages, diaphragm, the respiratory system, including nasal mucosa, trachea, larynx, pharynx, bronchi, lungs, the digestive system, including esophagus, stomach, intestines, tissues of the anal canal, tissues and mucous membranes of the oral cavity, including gums, teeth, tongue, the urinary system, including kidneys, ureters, bladder, urethra, adrenal glands, or the genital and reproductive system, including corpora cavernosa, prostate, uterus, vulva, vagina, endometrium, fallopian tubes. 
     
     
         44 . A method of treating bladder cancer, said method comprising administering the nanofibers according to  claim 1  or a hydrogel containing said nanofibers. 
     
     
         45 . The method according to  claim 40 , wherein said nanofibers are in a hydrogel system and said system comprises Mitomycin C as an active ingredient.